PROJECT SUMMARY More than 100 millions of people suffer from daily chronic pain worldwide. Opioids remain the mostly prescribed medications to treat chronic pain. However, long-term use of opioids causes paradoxical hyperalgesia and in addition, opioids produce strong rewarding effect and can cause opioid abuse and addiction, which may have contributed to the current opioid epidemic. Due to these severe side effects of opioids, the analgesic efficiency and clinical benefits of long-term opioids for chronic pain have been increasingly questioned in recent years. Therefore, there is an urgent need for new drugs or therapeutic options to treat chronic pain, which requires better understanding of chronic pain-induced changes in the brain without opioids and in the context of long-term opioids. However, it is still unclear what changes in the brain that mediate the development of chronic pain from acute pain and how chronic pain may change responses to opioid reward for the altered liability of opioid abuse under chronic pain. Our preliminary studies have found that Dnmt3a, a DNA methyltransferase that catalyzes DNA methylation for gene repression, is significantly downregulated in the brain in a time-dependent manner during the development of chronic pain. Moreover, repeated opioid treatment also downregulates Dnmt3a. Therefore, this proposal will investigate whether Dnmt3a acts as a key protein in the brain for the development of chronic pain and whether Dnmt3a could be a novel epigenetic target for the development of new drugs and therapeutic options for the treatment of chronic pain. We propose three specific aims to test our central hypothesis that persistent pain induces adaptive Dnmt3a decrease in key pain-modulating brain regions, which results in DNA hypomethylation and aberrant neuronal hyperactivity, leading to the development of chronic pain and increased sensitivity to opioid reward. Aim 1 will identify Dnmt3a as a key epigenetic protein in three representative pain-modulating brain regions for the development of chronic pain. Aim 2 will manipulate Dnmt3a levels in the brain regions to validate Dnmt3a as a potential drug target for the treatment of chronic pain. Aim 3 will identify any abuse liability of Dnmt3a upregulation as a treatment option and how it affects the rewarding effect of opioids for opioid abuse. These studies are expected to reveal critical roles of brain Dnmt3a in the development of chronic pain and provide fundamental insight for Dnmt3a as the target of novel therapeutic options that inhibit chronic pain development while decreasing abuse liability of opioids.